Double stranded RNA dependent protein kinase (PKR) is a member of a family of kinases that phosphorylates the alpha subunit of protein synthesis initiation factor, eIF-2 (eIF-2α) and plays a role in the translational down regulation of gene expression (Clemens et al. Mol. Biol. Rep. 1994; vol. 19: 210-10). Activation of PKR involves two molecules binding in tandem to double stranded RNA and then phosphorylating each other in an intramolecular event. (Wu et al. 1997, J. Biol. Chem 272:1291-1296). PKR has been implicated in processes that rely on apoptosis as control mechanisms in vivo including antiviral activities, cell growth regulation and tumorigenesis (Donze et al. EMBO J. 1995, vol. 14: 3828-34; Lee et al. Virology 1994, vol. 199: 491-6; Jagus et al. Int. J. Biochem. Cell. Biol. 1989, vol. 9: 1576-86).
The alpha subunit of protein synthesis initiation factor is responsible for binding the initiating methionyl-tRNA (Met-tRNAf), together with a molecule of GTP, and placing Met-tRNAf on native 40S ribosomal subunits (Pain, Eur. J. Biochem 1996, vol. 236; 747-771). During the course of this process GTP is hydrolyzed to GDP and inorganic phosphate, and when eIF-2 leaves the ribosome later in initiation (at the 60S subunit joining stage), it does so as an inactive eIF-2.GDP complex. Regeneration of active eIF-2 requires the exchange the GDP for a new molecule of GTP, catalyzed by the guanine nucleotide exchange factor eIF-2α (Pain, 1996). When eIF-2 becomes phosphorylated by PKR the initiation factor acquires an increased affinity for eIF-2α resulting in sequestration of the latter in an inactive complex. Consequently, the rate of guanine nucleotide exchange on both phosphorylated and unphosphorylated eIF-2 is decreased, as the concentration of available eIF-2α present in cell is reduced with respect to eIF-2, resulting in inhibition of polypeptide chain initiation. Not only does PKR activity have an effect on global rate of protein synthesis, but it may also selectively inhibit the translation of specific mRNAs that for various reasons have a greater than average requirement for active eIF-2 in the cell.
Regulation of protein synthesis through activated PKR arises from the interaction of PKR with double stranded RNA (dsRNA). The activation of PKR by dsRNA depends on the concentration and size of the dsRNA. In particular, PKR is activated by low levels of dsRNA and inhibited by higher levels of dsRNA. This gives rise to a characteristic bell-shaped curve for activation of the enzyme as a function of dsRNA concentration (Clemens et al., Proc. Natl. Acad. Sci. USA 1975; 72(4):1286-90; Levin D H. et al., Proc. Natl. Acad. Sci. USA 1980; 77(2):832-6 5). With respect to size of dsRNA, it has been found that molecules shorter than 30 base pairs (bp) in size fail to form a stable complex with PKR and do not activate the enzyme. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. Analysis of complexes between dsRNA and PKR suggests that at maximal packing, the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation, the binding site protects about 80 bp of duplex (Manche et al. Mol. Cell. Biol. 1992, vol. 12:5238-48).